A collaborative study to establish the national standard for SARS-CoV-2 RNA nucleic acid amplification techniques (NAAT) in Taiwan

Abstract

The conventional PCR remains a valuable method to detect the newly emergent coronavirus rapidly and accurately. Our investigation aimed to establish the standard materials of SARS-CoV-2 for NAAT detection. We provided formalin-inactivated SARS-CoV-2 and confirmed RNA copy numbers. In addition, the virus genome was confirmed with whole-genome sequencing and identified as Wuhan/WI04/2019. Seven laboratories were invited for this collaborative study, according to the reporting data, we determined the SARS-CoV-2 with the unit of 6.35 Log₁₀ copies/mL as the national standard. The availability of the national standard (NS) of SARS-CoV-2 will facilitate the standardization and harmonization of SARS-CoV-2 NAAT assays.

Keywords: International standard (IS); National standard (NS); Nucleic acid amplification techniques (NAAT); Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Fig. 1

The entire genome sequencing of SARS-CoV-2. The entire virus genome was sequenced, analyzed using Illumina Miseq., and compared with the reference genome (NC_045512.2 Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome). Eight nucleotides and two amino acid variants can be observed. Abbreviations: ORF = open reading frames; E = envelope glycoprotein gene; M = membrane glycoprotein gene; N = nucleocapsid gene.

Fig. 2

The preparation of inactivated SARS-CoV-2 national candidate standard was verified using three methods a) Vero E6 cells were inoculated with/without SARS-CoV-2 and formalin-inactivated virus. Significant CPE was observed in cells infected with the virus but not in the other two groups. b) Ct values quantitated by RT-qPCR with primer-probe targeting E and RdRp genes. After seven days of incubation, the supernatant was harvested and used to infect new prepared cells for another seven days for a total of three weeks. Error bars represent the standard deviation of duplicate reactions. Undetermined values were assigned a Ct value of 45. c) A total of 20 μg of protein were used to detect spike and nucleocapsid proteins. Protein lysate of 0.1 M.O.I was harvested on third day, while lysate of 0.001 MOI was harvested seven days after infection.

Fig. 3

Distribution chart of the quantitative results of the SARS-CoV-2 NS. The mean estimates from the individual laboratories for the SARS-CoV-2 NS were obtained using quantitative NAAT assays. The upper blue line is the mean + 2SD; the lower yellow line is the mean value -2SD. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

The stability assessment of the SARS-CoV-2 national standard. a) The SARS-CoV-2 national standard was stored at four temperatures: 24 °C, 4 °C, −20 °C, and −80 °C at specified time points. b) The RNA concentration of four temperatures were evaluated at corresponding times using RT-qPCR.